15-KETE对大鼠肺动脉平滑肌细胞钾离子通道的作用

Effect of 15-KETE on Voltage-Gated K^+ Current in Rat Intrapulmonary Arterial Smooth Muscle Cells

目的观察15-酮基二十碳四烯酸(15-ketoeicosatetraenoic acid,15-KETE)对大鼠肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)膜电压门控钾离子通道(Kv)的作用,探讨其收缩肺动脉的离子通道机制。方法采用急性酶分离法(胶原酶Ⅰ型和弹性酶)获得健康成年SD大鼠单个PASMC,应用全细胞膜片钳记录方法,研究15-KETE对膜电位(Em)、膜电容(Cm)、电压门控钾电流(Ikv)的影响。结果①高浓度15-KETE(1×10-7mol/L、1×10-6mol/L)可引起PASMCs去极化,并且在细胞内钙被BAPTA缓冲后,15-KETE仍可引起PASMCs去极化,15-KETE对PASMC的膜电容无影响;②15-KETE(1×10-8~1×10-6mol/L)对Ikv的影响呈浓度依赖性和可逆性;③细胞内钙离子在生理浓度时([Ca2+]i=75 nmol/L),15-KETE(1×10-6mol/L)对Ikv峰电流的抑制率显著高于细胞内无钙离子时。结论15-KETE可浓度依赖性的抑制Ikv,使常氧大鼠PASMCs去极化;细胞内钙离子加强了15-KETE对Ikv峰电流的抑制作用。

Objective To observe the effect of 15-KETE on voltage-gated K^＋ （Kv） channel in rat intrapulmonary arterial smooth muscle cells （PASMCs）, and investigate its contractile mechanisms on rat pulmonary artery. Methods Single PASMCs were obtained with acute enzyme （collagenase I plus elastase） dispersing method. Using whole cell patch-clamp technique in freshly isolated rat PASMCs, the effect of 15-KETE on Kv current was recorded. Results （1） 15-KETE（ 1 x 10-7 mol/L, 1 x 10-6 mol/L） caused membrane depolarization. The effect of 15-KETE on membrane potential persisted in cells in which intracellular Ca2 ~ was buffered with 1,2-bis （2-aminophenoxy） ethane-N, N, N＇, N＇-tetraacetic acid （ BAPTA）. 15-KETE did not affect the membrane capacitance of PASMCs. （2） 15-KETE （1 × 10^-8 to 1 × 10^-6 tool/L） caused a concentration-dependent and reversible inhibition of Kv current. （3） The inhibition of 15-KETE （1× 10^-6 mol/L） on peak Kv current was greater under conditions containing intraceUular Ca^2＋ at physiological level （[ Ca^2＋ ]i = 75nM） than that of free of intracellular Ca^2＋. Conclusion 15-KETE concentration-dependently inhibited the Kv current of PASMCs, and depolarized the membrane potential of PASMCs in normoxia rat; the mtracellular Ca strengthened the inhibition of 15-KETE on peak Kv current.